The kidney assumes a critical role in the maintenance of acid-base balance, extracellular fluid volume and blood pressure homeostasis via regulation of NaHCO3 and NaCI excretion. One protein that is central to all these processes is the Na/H exchanger isoform 3 (NHE3). NHE3 mediates a great majority of NaHCO3 and NaCI absorption and is regulated rapidly by hemodynamic and neurohumoral factors to match the demands of the organism. Recent work from several laboratories have shown that NHE3 can be acutely regulated via a number O mechanisms. We have shown that hormones such as parathyroid hormone (PTH) and dopamine (DA) acutely inhibits NHE3 transport activity without changing its protein abundance on the plasma membrane. This inhibition is associated with complex changes in NHE3 phosphorylation and clustering of NHE3 proteins. We hypothesize that changes in NHE3 phosphorylation leads to NHE3 clustering which results in inhibition of activity. In this proposal. we will focus on: 1. Mapping out the amino acid residues n NHE3 that is regulated. 2. Determine whether changes in NHE3 phosphorylation per se alters its activity. 3. Determine whether NHE3 clustering is a consequence of changes in NHE3 phosphorylation and whether NHE3 clustering alters its activity. 4. Determine the role of two NHE3 binding proteins in mediating NHE3 clustering and changes in phosphorylation. We will apply protocols in transport physiology, biochemistry, recombinant DNA technology, immunohistology, and biophysical fluorescence spectroscopy to study whole animals, culture cells, and purified proteins. These studies will uncover highly fundamental mechanisms of regulation of an important epithelial Na transporter and further our understanding 01 disturbance in acid-base balance, Na retention and hypertension.